Spatial Patterning of Molecular Cues and Vascular Cells in Fully Integrated Hydrogel Channels via Interfacial Bioorthogonal Cross-Linking

Abstract

Fully integrated hydrogel channels were fabricated via interfacial bioorthogonal crosslinking-a diffusion-controlled method for the creation and patterning of synthetic matrices based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) dienophiles. Injecting an aqueous solution of a bisTCO crosslinker into a reservoir of tetrazine-modified hyaluronic acid (HA-Tz), while simultaneously drawing the syringe needle through the reservoir, yielded a crosslinked hydrogel channel that was mechanically robust. Fluorescent tags and biochemical signals were spatially patterned in the channel wall through time-dependent perfusion of TCO-conjugated molecules into the lumen of the channel. Different cell populations were spatially encapsulated in the channel wall via temporal alteration of cells in the HA-Tz reservoir. The interfacial approach enabled spatial patterning of vascular cells, including human abdominal aorta endothelial cells, aortic vascular smooth muscle cells, and aortic adventitial fibroblasts, into the hydrogel channels with high viability and proper morphology in the anatomical order found in human arteries. The bioorthogonal platform does not rely on external triggers and represents a first step towards the engineering of functional and implantable arteries.

Enabling in vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Si-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts

“…Tetrazine ligation has been used for the creation of hydrogels for tissue engineering applications including in vivo cell delivery 87 and the creation of molecularly patterned matrices based on interfacial bioorthogonal chemistry. 88 Previously, Truong, Forsythe and coworkers used our first generation system (catalytic methylene blue, DHTz 2, 660 nm light) via crosslinking with a 4-arm PEG-norbornene to encapsulate human mesenchymal stem cells (hMSCs); however, cell viability beyond Day 1 was not described. 75 In our own experiments the phototoxicity of methylene blue and the sensitivity of DHTz 2 under cell culture conditions has limited the broader application of our first generation system for tissue engineering purposes.…”

Core–Shell Microfibers via Bioorthogonal Layer-by-Layer Assembly

“…To ensure rapid growth of the HA gel layer, the reaction should occur instantaneously under mild conditions. Tetrazine ligation, the inverse electron demand Diels–Alder cycloaddition reaction between a strained trans -cyclooctene (TCO) and s -tetrazine (Tz), is biocompatible, high yielding, and extremely fast (second-order rate constant, k 2 , exceeding 10 4 M –1 s –1 ) and does not require any catalyst, nor does it exhibit cross reactivity with endogenous biomacromolecules. − We have successfully applied this chemistry to the creation of protein-mimetic polymeric microfibers − and 3D biomimetic environments with well-defined spatiotemporal signals. − …”

“…Fiber surfaces were relatively smooth and free of any nanoscale topography. Separately, HA derivatives with bioorthogonal handles were synthesized following our reported procedures (Scheme S1 and Figure A). − By 1 H NMR, HA-Tz had a 23% tetrazine incorporation, and HA-TCO had a degree of modification of 25% (Figure S2). To introduce integrin-binding sites on the fibrous scaffold, the monofunctional TCO conjugate, RGD–TCO, was synthesized following our reported solid-phase synthesis protocol (Figures S3 and S4).…”

Advanced Bottom‐Up Engineering of Living Architectures

“…The researchers could then assemble heterogenous hydrogels with spatially resolved properties, such as stiff‐to‐soft transitions from shell to core, as well as increasing MMP degradability or adhesion site density toward the core. Recently, the same group has now devised multilayered hydrogel channels with three different cell populations spatially patterned across its interfaces, thus yielding functional bioarchitectures more closely resembling native arteries . Another relevant strategy to obtain cellularly graded hydrogels exploits the self‐healing phenomena that is characteristic of dynamic covalent hydrogel networks.…”